Tyrosinase inhibitors are important in cosmetic, medical, and food industries due to their regulation of melanin production. A tyrosinase inhibitor was purified from Camellia pollen using high-speed countercurrent chromatography and preparative high-performance liquid chromatography and was identified as caffeine by NMR and mass spectrometry. It showed strong mushroom tyrosinase inhibitory activity with an IC50 of 18.5 ± 2.31 μg/mL in a noncompetitive model. The caffeine did not interact with copper ions in the active center of the enzyme but could quench fluorescence intensity and change the secondary conformation of this tyrosinase. A molecular dynamics simulation showed that caffeine bound this tyrosinase via Lys379, Lys 376, Asp357, Glu356, Thr308, Gln307, Asp312, and Trp358, thus changing the binding sites of l-tyrosine and the loop conformation adjacent to the active center. In vitro cell model analysis revealed that caffeine exhibited significant inhibitory effects on both intracellular tyrosinase activity and melanin production of B16-F10 melanoma cells in a concentration-dependent manner. These comprehensive results suggest that caffeine is a strong tyrosinase inhibitor that has the potential to be developed as skin-whitening agents in the cosmetics and pharmaceutical industries or as antibrowning agents in the food industry.
Keywords: Camellia pollen; binding site; caffeine; noncompetitive inhibition; tyrosinase inhibition.